Identification of Host-Specific Bacteroidales 16S rDNA Sequences from Human Sewage and Ruminant Feces

The need to identify the source of fecal contamination of water has led to the development of various fecal source identification methods, a field known as microbial source tracking (MST). One promising method of MST focuses on fecal members of the order Bacteroidales, some of which exhibit a high degree of host-specificity. In order to identify host-specific Bacteroidales genetic markers, a ∼1060 bp section of Bacteroidales 16S rDNA was amplified from human sewage (n = 6), and bovine (n = 6) and ovine fecal (n = 5) samples and used for the generation of three clone libraries. Phylogenetic analysis of sequences from the three clone libraries revealed that the Bacteroidales species found in both human sewage and bovine and ovine feces were a highly diverse group of organisms, many of which were not represented by previously characterised 16S rDNA. Ovine and bovine feces appear to host similar populations of Bacteroidales species and these species were more diverse and less closely related to cultivated species than the Bacteroidales population found in human sewage. Species of Bacteroidales from the ruminant and human feces formed isolated clusters containing putatively host-specific sequences. These sequences were subsequently exploited for the design of host-specific primers which were used in MST studies

Bacterial community analysis on the Mediaeval stained glass window "Natività" in the Florence Cathedral

Microbial corrosion of glass causes problems on delicates antique glass samples. Until now, the effect of microbial activity on corrosion phenomena has not been well documented. Only a few studies have been published concerning the microflora growing on glass surfaces. The present study deals with the characterization of cultivable aerobic bacteria isolated from the historical glass window "Natività" in the Florence Cathedral, designed by Paolo Uccello and realized by Angelo Lippi between 1443 and 1444. Microbial strains were sampled from four of the 25 panels of the "Natività" in the occasion of a recent conservation treatment, due to the presence of various kinds of crusts. One hundred microorganisms were isolated, about 50% bacteria and 50% fungi. Bacteria were submitted to morphological characterization and classified in the Gram group. For twenty strains, from different glass panels, the 16S rDNA gene was amplified and sequenced. Sequence analysis showed genus Bacillus, Arthrobacter and Paenibacillus as the most representative. In particular Bacillus and Paenibacillus are crusts associated. Phylogenetic relationship among isolates was determined. Chemical analysis of the glass and crusts completed the study

Evaluating amplified rDNA restriction analysis assay for identification of bacterial communities

Amplified ribosomal DNA restriction analysis (ARDRA) and restriction fragment length polymorphism were originally used for strain typing and for screening clone libraries to identify phylogenetic clusters within a microbial community. Here we used ARDRA as a model to examine the capacity of restriction-based techniques for clone identification, and the possibility of deriving phylogenetic information from ARDRA-based dendrograms. ARDRA was performed in silico on 48,759 sequences from the Ribosomal Database Project, and it was found that the fragmentation profiles were not necessarily unique for each sequence in the database, resulting in different species sharing fragmentation profiles. Although ARDRA-based clusters separated clones into different genera, these phylogenetic clusters did not overlap with trees constructed according to sequence alignment, calling into question the intra-genus ARDRA-based phylogeny. It is thus suggested that the prediction power of ARDRA clusters in identifying clone phylogeny be regarded with caution

Changes in the bacterial community structure and diversity during bamboo retting

Microbial retting is a critical step in obtaining fiber bundles from bamboo culm using indigenous microorganisms. A cultivation-independent technique for monitoring the changes in bacteria community during bamboo retting was applied in this work. This technique involves genetic profiling of PCR-amplified small-subunit rRNA and the single-strand conformation polymorphism (SSCP) gel analysis of the PCR-amplified 16S rDNA fragments. The study revealed that both the structure and the diversity of investigated communities varied with the incubation periods and sample locations. The bacteria bands from SCCP gel profiles related to Bacillus sp. decreased in intensity, and Phaeospirillum sp. and Azospirillum brasilense completely disappeared during the 4th and 5th month of incubation, while the bands related to the Sphingomonas japonica, Alphaproteobacterium Ellin335 and Microbacterium sp. increased. The bands closely related to Sphingomonads, Brevundimonas brasilense, Pseudoclavibacter sp., Agrococcus jenensis and Oxalophagus oxalicus remained dominant during the whole incubation period. This study showed that the use of PCR assay targeting 16S rRNA and SCCP profiling provided valuable information on monitoring the bacteria dynamic changes occurring in the bacteria community during bamboo retting, which is crucial for controlling the quality of the retting process and improving the retting efficiency, and thus benefits for fiber recovery.This work was made possible by the support from the earmarked fund for Modern Agro-industry Technology Research System (nycytx-19-E23), the European Union Biorenew Project [Sixth Framework Programme (FP6-2004-NMP-NI-4)] and China Scholarship Council. The authors would like to thank Florian Schmid, Herbert Pobeheim, Stefan Wei\l=ss\, Michael Furnkranz, Christoph Schmidt and Endry Nugroho Prasetyo for a number of insightful comments and suggestions

Cultivation of a Synergistetes strain representing a previously uncultivated lineage

Subgingival plaque samples obtained from human subjects with periodontitis, shown to include previously uncultivable members of the phylum Synergistetes, were used to inoculate Cooked Meat Medium (CMM). The presence of Cluster A (uncultivable) Synergistetes was monitored by fluorescent in situ hybridization (FISH) and quantitative PCR (Q-PCR). Cluster A Synergistetes were found to grow in CMM in co-culture with other plaque bacteria and growth was stimulated by the addition of mucin and serum. Plaque samples were also used to inoculate Blood Agar (BA) plates and growth of Cluster A Synergistetes was revealed after anaerobic incubation, by colony hybridization with specific probes. Surface growth on the plates in regions identified by colony hybridization was harvested and used to inoculate fresh plates, thus enriching for Cluster A Synergistetes. Cross-streaks of other plaque bacteria were also used to stimulate Synergistetes growth. In the early passages, no discrete Synergistetes colonies were seen, but after eight passages and the use of cross-streaks of other bacteria present in the enriched community, colonies arose, which consisted solely of Cluster A Synergistetes cells, as determined by 16S rRNA gene PCR and cloning. This is the first report of the successful culture of a member of the uncultivable branch of this phylum

Dual and recombinant infections: an integral part of the HIV-1 epidemic in Brazil.

We systematically evaluated multiple and recombinant infections in an HIV-infected population selected for vaccine trials. Seventy-nine HIV-1 infected persons in a clinical cohort study in Rio de Janeiro, Brazil, were evaluated for 1 year. A combination of molecular screening assays and DNA sequencing showed 3 dual infections (3.8%), 6 recombinant infections (7.6%), and 70 (88.6%) infections involving single viral subtypes. In the three dual infections, we identified HIV-1 subtypes F and B, F and D, and B and D; in contrast, the single and recombinant infections involved only HIV-1 subtypes B and F. The recombinants had five distinct B/F mosaic patterns: Bgag-p17/Bgag-p24/Fpol/Benv, Fgag-p17/Bgag-p24/Fpol/Fenv, Bgag-p17/B-Fgag-p24/Fpol/Fenv, Bgag-p17/B-Fgag-p24/Fpol/Benv, and Fgag-p17/B-Fgag-p24/Fpol/Fenv. No association was found between dual or recombinant infections and demographic or clinical variables. These findings indicate that dual and recombinant infections are emerging as an integral part of the HIV/AIDS epidemic in Brazil and emphasize the heterogenous character of epidemics emerging in countries where multiple viral subtypes coexist

PCR-mediated detection of acidophilic, bioleaching-associated bacteria

The detection of acidophilic microorganisms from mining environments by culture methods is time consuming and unreliable. Several PCR approaches were developed to amplify small-subunit rRNA sequences from the DNA of six bacterial phylotypes associated with acidic mining environments, permitting the detection of the target DNA at concentrations as low as 10 fg